In recent years, there have been developed various methods for the diagnosis of diseases by detection of associated genes from bacteria, viruses or other pathogens. Some samples may contain nucleic acids in sufficient amounts for their direct detection. In contrast, when a target gene is present in a very small amount or proportion, the direct detection of the target gene becomes quite difficult. This problem has hitherto been solved with a technique of amplifying the target gene by cell or bacterial culture methods; however, these methods have a defect that they require much time.
For nucleic acid amplification, the polymerase chain reaction (PCR) method has also been known. In this method, the degree of amplification of a target nucleic acid is controlled by the number of cycles. The amplification rate is calculated by 2.sup.n where n is the number of cycles. The amplification of a target nucleic acid up to the amount making possible its actual detection requires 25 to 30 cycles.
Different methods for nucleic acid amplification based on RNA replication are disclosed in JP-A 2-5864, JP-A 2-500565 and JP-A 2-501532. In these methods, a promoter sequence for DNA-dependent RNA polymerase is incorporated into a primer for use in the synthesis of double-stranded DNA from a target nucleic acid. After the synthesis of double-stranded DNA, the resulting double-stranded DNA is used as a template to synthesize RNA corresponding to the target nucleic acid with DNA-dependent RNA polymerase.
The resulting RNA is then used for the synthesis of a DNA/RNA hybrid with RNA-dependent DNA polymerase, from which DNA/RNA hybrid the RNA is separated to yield single-stranded DNA. The DNA separation is achieved by heat denaturation (JP-A 2-500565 and JP-A 2-501532) or by use of ribonuclease H (JP-A 2-5864).
The single-stranded DNA thus obtained is used together with another primer to synthesize double-stranded DNA containing a promoter sequence for DNA-dependent RNA polymerase, and the resulting double-stranded DNA is subjected to RNA transcription.
The use of this method makes it possible to transcribe tens or thousands of RNA molecules from only one double-stranded nucleic acid molecule by DNA-dependent RNA polymerase, resulting in a higher efficiency of amplification per cycle as compared with the PCR method. In the case where ribonuclease H is used, temperature cycles, required in the PCR method, become unnecessary, and amplification can, therefore, be carried out in a simpler manner.
The amplification methods based on RNA replication have high efficiency of amplification; however, the conventional enzymes used in the respective reactions, such as RNA-dependent DNA polymerase, DNA-dependent RNA polymerase and DNA-dependent DNA polymerase, are usually non-thermostable. Low thermostability of these enzymes prevents the use of high temperatures in the reactions of amplification; therefore, non-specific hybridization between the template nucleic acid and the primer cannot be avoided, resulting in a decrease of specificity, which may cause a deterioration in the sensitivity of detection. To solve this problem, there has been developed a method with thermostable enzymes derived from Thermus thermophilus as the enzymes to be used in these amplification methods (JP-A 7-203999). In this method, the reactions of amplification are effected at a constant high temperature by use of thermostable enzymes, thereby making it possible to avoid non-specific hybridization.